生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 209-218.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0234
蒋晶晶1,3(), 周昭旭1,3, 杜蕙1,3, 吕昭龙2, 王春明1,3, 郭建国1,3, 张新瑞1,3, 李继平1,2,3()
收稿日期:
2023-03-16
出版日期:
2023-10-26
发布日期:
2023-11-28
通讯作者:
李继平,男,研究员,研究方向:经济作物病害及微生物资源;E-mail: lijiping@gsagr.ac.cn作者简介:
蒋晶晶,女,助理研究员,研究方向:经济作物病害病理学及生物防治;E-mail: jingjingziyu@163.com
基金资助:
JIANG Jing-jing1,3(), ZHOU Zhao-xu1,3, DU Hui1,3, LYU Zhao-long2, WANG Chun-ming1,3, GUO Jian-guo1,3, ZHANG Xin-rui1,3, LI Ji-ping1,2,3()
Received:
2023-03-16
Published:
2023-10-26
Online:
2023-11-28
摘要:
明确甘肃省静宁县及榆中县苹果褐腐病的病原种类,并筛选优质病原拮抗菌,为苹果采后病害的生物防治提供资源。采用常规组织分离法对苹果褐腐病病果进行病原菌分离纯化、柯赫氏法则验证、分子生物学方法结合形态学鉴定;采用平板对峙法筛选拮抗细菌,利用形态学、生理生化和分子生物学方法对拮抗菌进行鉴定,用离体果实测定拮抗菌防病效果。结果表明,引起甘肃静宁县及榆中县苹果褐腐病的病原菌为云南链核盘菌(Monilia yunnanensis),筛选获得的最优拮抗细菌X2-2为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),对M. yunnanensis的菌丝抑制率达(81.29±0.57)%,在离体果实上拮抗菌的防治效果达98.8%,能够显著抑制苹果褐腐病病斑在果实上的扩展速度。云南链核盘菌为甘肃部分地区苹果褐腐病主要病原菌,该研究筛选出的拮抗细菌X2-2抑菌效果显著,具有作为优质生防菌株的潜力。
蒋晶晶, 周昭旭, 杜蕙, 吕昭龙, 王春明, 郭建国, 张新瑞, 李继平. 甘肃部分地区苹果褐腐病病原分离鉴定及拮抗细菌筛选[J]. 生物技术通报, 2023, 39(10): 209-218.
JIANG Jing-jing, ZHOU Zhao-xu, DU Hui, LYU Zhao-long, WANG Chun-ming, GUO Jian-guo, ZHANG Xin-rui, LI Ji-ping. Isolation and Identification of Apple Brown Rot Pathogen in Parts of Gansu and Screening of Antagonistic Bacteria[J]. Biotechnology Bulletin, 2023, 39(10): 209-218.
图1 苹果褐腐病果实自然发病症状及病原菌接种症状 A-B:病害症状(A:‘秦冠’,B:‘红富士’);C,E:菌饼接种5 d(C:‘秦冠’,E:‘红富士’);D,F:孢子悬浮液接种5 d(D:‘秦冠’,F:‘红富士’)
Fig. 1 Disease characteristics of brown rot of apple and symptoms of pathogenic inoculation A-B: Disease symptom(A: ‘Qinguan apple’. B: ‘Red Fuji’ apple). C, E: Fungus cake inoculation for 5 d(C: ‘Qinguan’ apple. E: ‘Red Fuji’ apple). D, F: Spore suspension inoculation for 5 d(D: ‘Qinguan’ apple. F: ‘Red Fuji’ apple)
图2 苹果褐腐病菌的菌落形态及分生孢子形态 A:菌落正面;B:菌落背面;C:分生孢子梗(10×40);D:分生孢子(10×40);标尺=20 μm
Fig. 2 Colony and conidial morphology of apple brown rot bacteria A: The front of the colony. B: The back of the colony. C: Conidiophores(10×40). D: The conidium(10×40). Scale bar=20 μm
菌株编号 Strain No. | 抑菌率 Inhibition rate/% | 抑菌带 Inhibition bands/mm |
---|---|---|
X2-2 | 81.29±0.57a | 7.42±0.17a |
Zyx-3 | 80.08±0.66b | 5.42±0.51a |
X1-5 | 74.64±0.97c | 4.25±0.38a |
X1-6 | 71.29±0.62d | 4.11±0.42a |
P-sy-3 | 70.88±0.52d | 3.20±0.55a |
表1 拮抗菌对苹果褐腐病菌的抑制作用
Table 1 Inhibition effect of antagonistic bacterium on M. yunnanensis
菌株编号 Strain No. | 抑菌率 Inhibition rate/% | 抑菌带 Inhibition bands/mm |
---|---|---|
X2-2 | 81.29±0.57a | 7.42±0.17a |
Zyx-3 | 80.08±0.66b | 5.42±0.51a |
X1-5 | 74.64±0.97c | 4.25±0.38a |
X1-6 | 71.29±0.62d | 4.11±0.42a |
P-sy-3 | 70.88±0.52d | 3.20±0.55a |
图5 接种时间对病斑面积和防治效果的影响 A:先接菌饼,24 h后接X2-2菌悬液;B:菌饼+菌悬液;C:先接X2-2菌悬液,24 h后接菌饼。图中小写字母表示在P<0.05水平差异显著
Fig. 5 Effect of inoculation time on lesion area and control effect A: Inoculated with the M. yunnanensis first, and then inoculated with the X2-2 bacterial suspension after 24 h. B: M. yunnanensis + bacterial suspension. C: First inoculated with X2-2 bacterial suspension, then inoculated with M. yunnanensis after 24 h. Lowercase letters indicate significant difference at P<0.05 level
图6 X2-2菌悬液对苹果褐腐病病斑扩展的抑制效果 A:先接菌饼,24 h后接X2-2菌悬液;B:菌饼+菌悬液;C:先接X2-2菌悬液,24 h后接菌饼
Fig. 6 Inhibition effect of X2-2 bacterial suspension on the lesion expansion of M. yunnanensis A: Inoculated with the M. yunnanensis first, and then inoculated with the X2-2 bacterial suspension after 24 h. B: M. yunnanensis + bacterial suspension. C: First inoculated with X2-2 bacterial suspension, then inoculated with M. yunnanensis after 24 h
指标 Index | 结果 Result | 指标 Index | 结果 Result | |
---|---|---|---|---|
革兰氏染色 Gram staining | + | V.P试验 V.P test | + | |
淀粉水解 Amylolysis | + | 甲基红试验 Methyl red test | + | |
明胶液化 Gelatin liquefaction | + | 柠檬酸盐 Citrate utilization | + | |
酪蛋白水解 Caseinhydrolyzation | - | 硫化氢试验 Hydrogen sulfide test | - | |
7% NaCl | + | 接触酶 Catalase | + |
表2 拮抗菌X2-2的生理生化鉴定结果
Table 2 Physiological and biochemical identification results of antagonistic bacterium X2-2
指标 Index | 结果 Result | 指标 Index | 结果 Result | |
---|---|---|---|---|
革兰氏染色 Gram staining | + | V.P试验 V.P test | + | |
淀粉水解 Amylolysis | + | 甲基红试验 Methyl red test | + | |
明胶液化 Gelatin liquefaction | + | 柠檬酸盐 Citrate utilization | + | |
酪蛋白水解 Caseinhydrolyzation | - | 硫化氢试验 Hydrogen sulfide test | - | |
7% NaCl | + | 接触酶 Catalase | + |
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